An Early Treatment With BKI-1748 Exhibits Full Protection Against Abortion and Congenital Infection in Sheep Experimentally Infected With Toxoplasma gondii.

Congenital toxoplasmosis in humans and in other mammalian species, such as small ruminants, is a well-known cause of abortion and fetal malformations. The calcium-dependent protein kinase 1 (CDPK1) inhibitor BKI-1748 has shown a promising safety profile for its use in humans and a good efficacy against Toxoplasma gondii infection in vitro and in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg did not show systemic or pregnancy-related toxicity. In sheep experimentally infected at 90 days of pregnancy with 1000 TgShSp1 oocysts, the BKI-1748 treatment administered from 48 hours after infection led to complete protection against abortion and congenital infection. In addition, compared to infected/untreated sheep, treated sheep showed a drastically lower rectal temperature increase and none showed IgG seroconversion throughout the study. In conclusion, BKI-1748 treatment in pregnant sheep starting at 48 hours after infection was fully effective against congenital toxoplasmosis.

Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

Recent advances on the development of bumped kinase inhibitors for treatment of cryptosporidiosis have focused on the 5-aminopyrazole-4-carboxamide scaffold, due to analogs that have less hERG inhibition, superior efficacy, and strong in vitro safety profiles. Three compounds, BKI-1770, -1841, and -1708, showed strong efficacy in C. parvum infected mice. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Appraisal of Fungus-Derived Xanthoquinodins as Broad-Spectrum Anti-Infectives Targeting Phylogenetically Diverse Human Pathogens.

Xanthoquinodins make up a distinctive class of xanthone-anthraquinone heterodimers reported as secondary metabolites from several fungal species. Through a collaborative multi-institutional screening program, a fungal extract prepared from a Trichocladium sp. was identified that exhibited strong inhibitory effects against several human pathogens (Mycoplasma genitalium, Plasmodium falciparum, Cryptosporidium parvum, and Trichomonas vaginalis). This report focuses on one of the unique samples that exhibited a desirable combination of biological effects: namely, it inhibited all four test pathogens and demonstrated low levels of toxicity toward HepG2 (human liver) cells. Fractionation and purification of the bioactive components and their congeners led to the identification of six new compounds [xanthoquinodins NPDG A1-A5 (1-5) and B1 (6)] as well as several previously reported natural products (7-14). The chemical structures of 1-14 were determined based on interpretation of their 1D and 2D NMR, HRESIMS, and electronic circular dichroism (ECD) data. Biological testing of the purified metabolites revealed that they possessed widely varying levels of inhibitory activity against a panel of human pathogens. Xanthoquinodins A1 (7) and A2 (8) exhibited the most promising broad-spectrum inhibitory effects against M. genitalium (EC50 values: 0.13 and 0.12 µM, respectively), C. parvum (EC50 values: 5.2 and 3.5 µM, respectively), T. vaginalis (EC50 values: 3.9 and 6.8 µM, respectively), and P. falciparum (EC50 values: 0.29 and 0.50 µM, respectively) with no cytotoxicity detected at the highest concentration tested (HepG2 EC50 > 25 µM).

Repurposing the Kinase Inhibitor Mavelertinib for Giardiasis Therapy.

A phenotypic screen of the ReFRAME compound library was performed to identify cell-active inhibitors that could be developed as therapeutics for giardiasis. A primary screen against Giardia lamblia GS clone H7 identified 85 cell-active compounds at a hit rate of 0.72%. A cytotoxicity counterscreen against HEK293T cells was carried out to assess hit compound selectivity for further prioritization. Mavelertinib (PF-06747775), a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), was identified as a potential new therapeutic based on indication, activity, and availability after reconfirmation. Mavelertinib has in vitro efficacy against metronidazole-resistant 713-M3 strains. Other EGFR-TKIs screened in follow-up assays exhibited insignificant inhibition of G. lamblia at 5 µM, suggesting that the primary molecular target of mavelertinib may have a different mechanistic binding mode from human EGFR-tyrosine kinase. Mavelertinib, dosed as low as 5 mg/kg of body weight or as high as 50 mg/kg, was efficacious in the acute murine Giardia infection model. These results suggest that mavelertinib merits consideration for repurposing and advancement to giardiasis clinical trials while its analogues are further developed.

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase (PfKRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both PfKRS1 and C. parvum KRS (CpKRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between PfKRS1 and CpKRS. This series of compounds inhibit CpKRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for PfKRS1 and CpKRS vs. (human) HsKRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.

Taming the Boys for Global Good: Contraceptive Strategy to Stop Malaria Transmission.

Transmission of human malaria parasites (Plasmodium spp.) by Anopheles mosquitoes is a continuous process that presents a formidable challenge for effective control of the disease. Infectious gametocytes continue to circulate in humans for up to four weeks after antimalarial drug treatment, permitting prolonged transmission to mosquitoes even after clinical cure. Almost all reported malaria cases are transmitted to humans by mosquitoes, and therefore decreasing the rate of Plasmodium transmission from humans to mosquitoes with novel transmission-blocking remedies would be an important complement to other interventions in reducing malaria incidence.

P-Glycoprotein-Mediated Efflux Reduces the In Vivo Efficacy of a Therapeutic Targeting the Gastrointestinal Parasite Cryptosporidium.

Recent studies have illustrated the burden Cryptosporidium infection places on the lives of malnourished children and immunocompromised individuals. Treatment options remain limited, and efforts to develop a new therapeutic are currently underway. However, there are unresolved questions about the ideal pharmacokinetic characteristics of new anti-Cryptosporidium therapeutics. Specifically, should drug developers optimize therapeutics and formulations to increase drug exposure in the gastrointestinal lumen, enterocytes, or systemic circulation? Furthermore, how should researchers interpret data suggesting their therapeutic is a drug efflux transporter substrate? In vivo drug transporter-mediated alterations in efficacy are well recognized in multiple disease areas, but the impact of intestinal transporters on therapeutic efficacy against enteric diseases has not been established. Using multiple in vitro models and a mouse model of Cryptosporidium infection, we characterized the effect of P-glycoprotein efflux on bumped kinase inhibitor pharmacokinetics and efficacy. Our results demonstrated P-glycoprotein decreases bumped kinase inhibitor enterocyte exposure, resulting in reduced in vivo efficacy against Cryptosporidium. Furthermore, a hollow fiber model of Cryptosporidium infection replicated the in vivo impact of P-glycoprotein on anti-Cryptosporidium efficacy. In conclusion, when optimizing drug candidates targeting the gastrointestinal epithelium or gastrointestinal epithelial infections, drug developers should consider the adverse impact of active efflux transporters on efficacy.

Pharmacokinetics and In Vivo Efficacy of Pyrazolopyrimidine, Pyrrolopyrimidine, and 5-Aminopyrazole-4-Carboxamide Bumped Kinase Inhibitors against Toxoplasmosis.

Bumped kinase inhibitors (BKIs) have been shown to be potent inhibitors of Toxoplasma gondii calcium-dependent protein kinase 1. Pyrazolopyrimidine and 5-aminopyrazole-4-carboxamide scaffold-based BKIs are effective in acute and chronic experimental models of toxoplasmosis. Through further exploration of these 2 scaffolds and a new pyrrolopyrimidine scaffold, additional compounds have been identified that are extremely effective against acute experimental toxoplasmosis. The in vivo efficacy of these BKIs demonstrates that the cyclopropyloxynaphthyl, cyclopropyloxyquinoline, and 2-ethoxyquinolin-6-yl substituents are associated with efficacy across scaffolds. In addition, a broad range of plasma concentrations after oral dosing resulted from small structural changes to the BKIs. These select BKIs include anti-Toxoplasma compounds that are effective against acute experimental toxoplasmosis and are not toxic in human cell assays, nor to mice when administered for therapy. The BKIs described here are promising late leads for improving anti-Toxoplasma therapy.

Treatment with Bumped Kinase Inhibitor 1294 Is Safe and Leads to Significant Protection against Abortion and Vertical Transmission in Sheep Experimentally Infected with Toxoplasma gondii during Pregnancy.

Previous studies on drug efficacy showed low protection against abortion and vertical transmission of Toxoplasma gondii in pregnant sheep. Bumped kinase inhibitors (BKIs), which are ATP-competitive inhibitors of calcium-dependent protein kinase 1 (CDPK1), were shown to be highly efficacious against several apicomplexan parasites in vitro and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 µM and trough concentrations of 0.4 µM at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a T. gondii oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation in vitro and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from T. gondii infection during pregnancy.

Optimization of Methionyl tRNA-Synthetase Inhibitors for Treatment of Cryptosporidium Infection.

Cryptosporidiosis is one of the leading causes of moderate to severe diarrhea in children in low-resource settings. The therapeutic options for cryptosporidiosis are limited to one drug, nitazoxanide, which unfortunately has poor activity in the most needy populations of malnourished children and HIV-infected persons. We describe here the discovery and early optimization of a class of imidazopyridine-containing compounds with potential for treating Cryptosporidium infections. The compounds target the Cryptosporidium methionyl-tRNA synthetase (MetRS), an enzyme that is essential for protein synthesis. The most potent compounds inhibited the enzyme with Ki values in the low picomolar range. Cryptosporidium cells in culture were potently inhibited with 50% effective concentrations as low as 7 nM and >1,000-fold selectivity over mammalian cells. A parasite persistence assay indicates that the compounds act by a parasiticidal mechanism. Several compounds were demonstrated to control infection in two murine models of cryptosporidiosis without evidence of toxicity. Pharmacological and physicochemical characteristics of compounds were investigated to determine properties that were associated with higher efficacy. The results indicate that MetRS inhibitors are excellent candidates for development for anticryptosporidiosis therapy.

Toxoplasma Calcium-Dependent Protein Kinase 1 Inhibitors: Probing Activity and Resistance Using Cellular Thermal Shift Assays.

In Toxoplasma gondii, calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC50s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC50s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC50s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC50s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.

Therapeutic Efficacy of Bumped Kinase Inhibitor 1369 in a Pig Model of Acute Diarrhea Caused by Cryptosporidium hominis.

Recent reports highlighting the global significance of cryptosporidiosis among children have renewed efforts to develop control measures. We evaluated the efficacy of bumped kinase inhibitor (BKI) 1369 in the gnotobiotic piglet model of acute diarrhea caused by Cryptosporidium hominis, the species responsible for most human cases. Five-day treatment with BKI 1369 reduced signs of disease early during treatment compared to those of untreated animals. Piglets treated with BKI 1369 exhibited significant reductions of oocyst excretion, mucosal colonization by C. hominis, and mucosal lesions, which resulted in considerable symptomatic improvement. BKI 1369 reduced the parasite burden and disease severity in the gnotobiotic pig model. Together these data suggest that a BKI-mediated therapeutic may be an effective treatment against cryptosporidiosis.

Necessity of Bumped Kinase Inhibitor Gastrointestinal Exposure in Treating Cryptosporidium Infection.

There is a substantial need for novel therapeutics to combat the widespread impact caused by Crytosporidium infection. However, there is a lack of knowledge as to which drug pharmacokinetic (PK) characteristics are key to generate an in vivo response, specifically whether systemic drug exposure is crucial for in vivo efficacy. To identify which PK properties are correlated with in vivo efficacy, we generated physiologically based PK models to simulate systemic and gastrointestinal drug concentrations for a series of bumped kinase inhibitors (BKIs) that have nearly identical in vitro potency against Cryptosporidium but display divergent PK properties. When BKI concentrations were used to predict in vivo efficacy with a neonatal model of Cryptosporidium infection, these concentrations in the large intestine were the sole predictors of the observed in vivo efficacy. The significance of large intestinal BKI exposure for predicting in vivo efficacy was further supported with an adult mouse model of Cryptosporidium infection. This study suggests that drug exposure in the large intestine is essential for generating a superior in vivo response, and that physiologically based PK models can assist in the prioritization of leading preclinical drug candidates for in vivo testing.

Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy.

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.

5-Aminopyrazole-4-Carboxamide-Based Compounds Prevent the Growth of Cryptosporidium parvum.

Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) is a promising target for drug development against cryptosporidiosis. We report a series of low-nanomolar CpCDPK1 5-aminopyrazole-4-carboxamide (AC) scaffold inhibitors that also potently inhibit C. parvum growth in vitro Correlation between anti-CpCDPK1 and C. parvum growth inhibition, as previously reported for pyrazolopyrimidines, was not apparent. Nonetheless, lead AC compounds exhibited a substantial reduction of parasite burden in the neonatal mouse cryptosporidiosis model when dosed at 25 mg/kg.

Development of a murine vertical transmission model for Toxoplasma gondii oocyst infection and studies on the efficacy of bumped kinase inhibitor (BKI)-1294 and the naphthoquinone buparvaquone against congenital toxoplasmosis.

Objectives: Establishment of a mouse model for congenital toxoplasmosis based on oral infection with oocysts from Toxoplasma gondii ME49 and its application for investigating chemotherapeutic options against congenital toxoplasmosis. Methods: CD1 mice were mated, orally infected with 5, 25, 100, 500 or 2000 oocysts and monitored for clinical signs and survival of dams and pups until 4 weeks post partum . The parasite burden in infected mice was quantified by real-time PCR in lungs, brains and, in the case of surviving pups, also in eyes. Seroconversion was assessed by ELISA. T. gondii cysts in brain were identified by immunofluorescence. In a second experiment, pregnant CD1 mice challenged with 20 oocysts/mouse were treated with buparvaquone or the calcium-dependent protein kinase 1 inhibitor bumped kinase inhibitor (BKI)-1294 and the outcome of infection was analysed. Results: T. gondii DNA was detected in the brain of all infected animals, irrespective of the infection dose. Seroconversion occurred at 3 weeks post-infection. Most pups born to infected dams died within 1 week post partum , but a small fraction survived until the end of the experiment. T. gondii DNA was detected in the brain of all survivors and half of them exhibited ocular infection. Chemotherapy with both compounds led to dramatically increased numbers of surviving pups and reduced cerebral infection. Most efficient were treatments with BKI-1294, with 100% survivors and only 7% brain-positive pups. Conclusions: BKI-1294 and buparvaquone exert excellent activities against transplacental transmission in pregnant mice.

Discovery of N-(2-aminoethyl)-N-benzyloxyphenyl benzamides: New potent Trypanosoma brucei inhibitors.

A phenotypic screen of a compound library for antiparasitic activity on Trypanosoma brucei, the causative agent of Human African Trypanosomiasis (HAT), led to the identification of N-(2-aminoethyl)-N-phenyl benzamides as a starting point for hit-to-lead medicinal chemistry. Eighty two analogues were prepared, which led to the identification of a set of highly potent N-(2-aminoethyl)-N-benzyloxyphenyl benzamides with the most potent compound 73 having an in vitro EC50=0.001µM. The compounds displayed drug-like properties when tested in a number of in vitro assays. Compound 73 was orally bioavailable and displayed good plasma and brain exposure in mice, cured 2 out of 3 mice infected with Trypanosoma brucei in acute model when dosed orally at 50mg/kg once per day for 4days. Given its potent antiparasitic properties and its ease of synthesis, compound 73 represents a potential lead for the development of drug to treat Human African Trypanosomiasis.

Extended-spectrum antiprotozoal bumped kinase inhibitors: A review.

Many life-cycle processes in parasites are regulated by protein phosphorylation. Hence, disruption of essential protein kinase function has been explored for therapy of parasitic diseases. However, the difficulty of inhibiting parasite protein kinases to the exclusion of host orthologues poses a practical challenge. A possible path around this difficulty is the use of bumped kinase inhibitors for targeting calcium-dependent protein kinases that contain atypically small gatekeeper residues and are crucial for pathogenic apicomplexan parasites' survival and proliferation. In this article, we review efficacy against the kinase target, parasite growth in vitro, and in animal infection models, as well as the relevant pharmacokinetic and safety parameters of bumped kinase inhibitors.

A Novel Calcium-Dependent Kinase Inhibitor, Bumped Kinase Inhibitor 1517, Cures Cryptosporidiosis in Immunosuppressed Mice.

Cryptosporidium is recognized as one of the main causes of childhood diarrhea worldwide. However, the current treatment for cryptosporidiosis is suboptimal. Calcium flux is essential for entry in apicomplexan parasites. Calcium-dependent protein kinases (CDPKs) are distinct from protein kinases of mammals, and the CDPK1 of the apicomplexan Cryptosporidium lack side chains that typically block a hydrophobic pocket in protein kinases. We exploited this to develop bumped kinase inhibitors (BKIs) that selectively target CDPK1. We have shown that several BKIs of Cryptosporidium CDPK1 potently reduce enzymatic activity and decrease parasite numbers when tested in vitro. In the present work, we studied the anticryptosporidial activity of BKI-1517, a novel BKI. The half maximal effective concentration for Cryptosporidium parvum in HCT-8 cells was determined to be approximately 50 nM. Silencing experiments of CDPK1 suggest that BKI-1517 acts on CDPK1 as its primary target. In a mouse model of chronic infection, 5 of 6 SCID/beige mice (83.3%) were cured after treatment with a single daily dose of 120 mg/kg BKI-1517. No side effects were observed. These data support advancing BKI-1517 as a lead compound for drug development for cryptosporidiosis.

Novel Bumped Kinase Inhibitors Are Safe and Effective Therapeutics in the Calf Clinical Model for Cryptosporidiosis.

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a diarrheal disease that has produced a large global burden in mortality and morbidity in humans and livestock. There are currently no consistently effective parasite-specific pharmaceuticals available for this disease. Bumped kinase inhibitors (BKIs) specific for parasite calcium-dependent protein kinases (CDPKs) have been shown to reduce infection in several parasites having medical and veterinary importance, including Toxoplasma gondii, Plasmodium falciparum, and C. parvum In the present study, BKIs were screened for efficacy against C. parvum infection in the neonatal mouse model. Three BKIs were then selected for safety and clinical efficacy evaluation in the calf model for cryptosporidiosis. Significant BKI treatment effects were observed for virtually all clinical and parasitological scoring parameters, including diarrhea severity, oocyst shedding, and overall health. These results provide proof of concept for BKIs as therapeutic drug leads in an animal model for human cryptosporidiosis.